This invention relates generally to a method for increasing the uptake of drugs into tumors by treatment with a nucleic acid ligand to PDGF in combination with a cytotoxic agent. The method used for identifying nucleic acid ligands to PDGF is called SELEX, an acronym for Systematic Evolution of Ligands by Exponential enrichment. The method of the present invention is useful for increasing the therapeutic effectiveness of cytotoxic agents.
The Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment,xe2x80x9d now abandoned, U.S. Pat. No. 5,475,096, entitled xe2x80x9cNucleic Acid Ligands,xe2x80x9d and U.S. Pat. No. 5,270,163 (see also WO 91/19813), entitled xe2x80x9cMethods for Identifying Nucleic Acid Ligands,xe2x80x9d each of which is specifically incorporated herein by reference in its entirety. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled xe2x80x9cMethod for Selecting Nucleic Acids on the Basis of Structure,xe2x80x9d describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled xe2x80x9cPhotoselection of Nucleic Acid Ligands,xe2x80x9d now abandoned, U.S. Pat. Nos. 5,763,177 and 6,011,577, both entitled xe2x80x9cSystematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,xe2x80x9d describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737, entitled xe2x80x9cHigh-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,xe2x80x9d describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,xe2x80x9d describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled xe2x80x9cHigh Affinity Nucleic Acid Ligands Containing Modified Nucleotides,xe2x80x9d that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2xe2x80x2-positions of pyrimidines. U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2xe2x80x2-amino (2xe2x80x2-NH2), 2xe2x80x2-fluoro (2xe2x80x2-F), and/or 2xe2x80x2-O-methyl (2xe2x80x2-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled xe2x80x9cNovel Method of Preparation of Known and Novel 2xe2x80x2 Modified Nucleosides by Intramolecular Nucleophilic Displacement,xe2x80x9d describes oligonucleotides containing various 2xe2x80x2-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,xe2x80x9d and U.S. Pat. No. 5,683,867, entitled xe2x80x9cSystematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,xe2x80x9d respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. Pat. No. 6,011,020, entitled xe2x80x9cNucleic Acid Ligand Complexes.xe2x80x9d Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
One approach to increasing the effectiveness of existing anti-cancer drugs for the treatment of solid malignancies is to augment the uptake of the drugs into tumors, and thereby obtain increased therapeutic concentration without elevating the adverse side-effects. Most solid tumors display an increased interstitial fluid pressure (IFP). The molecular mechanisms causing increased tumor IFP are poorly understood. However, tumor stroma involvement in IFP control has been demonstrated. (Gullino et al. (1964) Cancer Res. 24:780-797; Philips et al. (1990) J. Natl Cancer Inst. 82:1457-1469; Jain (1987) Cancer Res. 47:3039-3051). It has been suggested that high tumor IFP prevents drug transport from the circulation into the tumor interstitium. The reduction of tumor IFP therefore is a target for efforts to increase tumor drug uptake. (Jain (1996) Science 271:1079-1080). Several agents which induce a lowering of IFP, and thereby increase the transcapillary transport in experimental murine tumors have been identified, including nicotinamide (Lee et al. (1992) Cancer Res. 52:3237-3240), TNF-xcex1 (Kristensen et al. (1996) Br. J. Cancer 74:533-536) and dexamethasone (Kristjansen et al. (1993) Cancer Res. 53:4764-4766).
Platelet-derived growth factor (PDGF) and the cognate tyrosine kinase receptors are potent mitogens for mesenchymal cells. In addition to its growth promoting effects, PDGF-BB is involved also in the regulation of IFP. After dextran-induced anaphylaxis and lowering of IFP in rat skin, local administration of PDGF-BB results in normalized IFP. PDGF receptors are expressed in the tumor stroma of many common solid tumors, e.g. lung, colon and breast carcinomas. Based on these observations the effects of PDGF antagonists on tumor IFP, tumor transcapillary transport and therapeutic effects of cytotoxic drugs were investigated.
The present invention includes a method for treating tumors, more specifically, solid tumors comprising administering to a host a therapeutically effective dose of a composition comprising a PDGF aptamer and a cytotoxic agent. In a preferred embodiment the PDGF aptamer is identified using the SELEX process for the Systematic Evolution of Ligands by EXponential enrichment. The present invention also includes a method for reducing the interstitial fluid pressure (IFP) of a tumor, more specifically, a solid tumor comprising administering a PDGF nucleic acid ligand. Finally, the present invention includes a method for increasing the uptake of cytotoxic agents into a tumor comprising administering to a host a composition comprising a PDGF aptamer and a cytotoxic agent. The present invention provides a novel method to increase drug uptake and therefore the therapeutic effectiveness of chemotherapy, by treatment with a PDGF inhibitor in conjunction with the therapeutic agent. As illustrated below, treatment with a PDGF aptamer decreases interstitial hypertension in these tumors thereby increasing the uptake of the therapeutic agent.